Human Coagulation Factor VII Variants

ABSTRACT

The present invention encompasses isolated human coagulation Factor VII variants comprising a substitution of Phe in position 374 of SEQ ID NO 1 with another amino acid residue.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of Ser. No. 12/178,280 filed Jul. 23,2008 which is a continuation of Ser. No. 11/111,072 filed Apr. 21, 2005which is a continuation of Ser. No. 09/848,107 filed May 3, 2001 andclaims the benefit of priority under 35 U.S.C. 119 of Danish applicationno. PA 2000 00734 filed on May 3, 2000, Danish application no. PA 200001360 filed on Sep. 13, 2000, U.S. provisional application No.60/204,712 filed on May 16, 2000, and U.S. provisional application No.60/236,892 filed on Sep. 29, 2000, the contents of which are fullyincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to novel human coagulation Factor VIIavariants having coagulant activity as well as nucleic acid constructsencoding such variants, vectors and host cells comprising and expressingthe nucleic acid, pharmaceutical compositions, uses and methods oftreatment.

BACKGROUND OF THE INVENTION

Blood coagulation is a process consisting of a complex interaction ofvarious blood components (or factors) that eventually gives raise to afibrin clot. Generally, the blood components, which participate in whathas been referred to as the coagulation “cascade”, are enzymaticallyinactive proteins (proenzymes or zymogens) that are converted toproteolytic enzymes by the action of an activator (which itself is anactivated clotting factor). Coagulation factors that have undergone sucha conversion are generally referred to as “active factors”, and aredesignated by the addition of the letter “a” to the name of thecoagulation factor (e.g. Factor VIIa).

Initiation of the haemostatic process is mediated by the formation of acomplex between tissue factor, exposed as a result of injury to thevessel wall, and Factor VIIa. This complex then converts Factors IX andX to their active forms. Factor Xa converts limited amounts ofprothrombin to thrombin on the tissue factor-bearing cell. Thrombinactivates platelets and Factors V and VIII into Factors Va and VIIIa,both cofactors in the further process leading to the full thrombinburst. This process includes generation of Factor Xa by Factor IXa (incomplex with factor VIIIa) and occurs on the surface of activatedplatelets. Thrombin finally converts fibrinogen to fibrin resulting information of a fibrin clot. In recent years Factor VII and tissue factorhave been found to be the main initiators of blood coagulation.

Factor VII is a trace plasma glycoprotein that circulates in blood as asingle-chain zymogen. The zymogen is catalytically inactive.Single-chain Factor VII may be converted to two-chain Factor VIIa byFactor Xa, Factor XIIa, Factor IXa, Factor VIIa or thrombin in vitro.Factor Xa is believed to be the major physiological activator of FactorVII. Like several other plasma proteins involved in haemostasis, FactorVII is dependent on Vitamin K for its activity, which is required forthe gamma-carboxylation of multiple glutamic acid residues that areclustered close to the amino terminus of the protein. Thesegamma-carboxylated glutamic acids are required for the metal ion-inducedinteraction of Factor VII with phospholipids. The conversion of zymogenFactor VII into the activated two-chain molecule occurs by cleavage ofan internal Arg₁₅₂-Ile₁₅₃ peptide bond. In the presence of tissuefactor, phospholipids and calcium ions, the two-chain Factor VIIarapidly activates Factor X or Factor IX by limited proteolysis.

It is often desirable to stimulate or improve the coagulation cascade ina subject. Factor VIIa has been used to control bleeding disorders thathave several causes such as clotting factor deficiencies (e.g.haemophilia A and B or deficiency of coagulation Factors XI or VII) orclotting factor inhibitors. Factor VIIa has also been used to controlexcessive bleeding occurring in subjects with a normally functioningblood clotting cascade (no clotting factor deficiencies or inhibitorsagainst any of the coagulation factors). Such bleeding may, for example,be caused by a defective platelet function, thrombocytopenia or vonWillebrand's disease. Bleeding is also a major problem in connectionwith surgery and other forms of tissue damage.

European Patent No. 200,421 (ZymoGenetics) relates to the nucleotidesequence encoding human Factor VII and the recombinant expression ofFactor VII in mammalian cells.

Dickinson et al. (Proc. Natl. Acad. Sci. USA (1996) 93, 14379-14384)relates to a Factor VII variant wherein Leu305 has been replaced by Ala(FVII(Ala305)).

Iwanaga et al. (Thromb. Haemost. (supplement August 1999), 466, abstract1474) relates to Factor VIIa variants wherein residues 316-320 aredeleted or residues 311-322 are replaced with the corresponding residuesfrom trypsin.

There is, however, still a need for variants of Factor VIIa havingcoagulant activity, variants with high activity that can be administeredat relatively low doses, and variants which do not produce theundesirable side effects such as systemic activation of the coagulationsystem and bleeding, respectively, associated with conventionaltherapies.

SUMMARY OF THE INVENTION

The invention provides coagulation Factor VIIa variants with coagulantactivity. In a first aspect, the invention provides a human coagulationFactor VII variant, wherein the Leu residue in position 305 or the Pheresidue in position 374 of SEQ ID NO 1 has been replaced by anotheramino acid residue which can be encoded by nucleic acid constructs and,optionally, wherein at least one other amino acid residue in theremaining positions in the protease domain has been replaced by anotheramino acid residue which can be encoded by nucleic acid constructs; withthe proviso that the variant is not FVII(Ala305).

In one embodiment, the Leu residue in position 305 or the Phe residue inposition 374 of SEQ ID NO 1 and at the most 20 amino acid residues inthe remaining positions in the protease domain (positions 153-406) havebeen replaced. In one embodiment, at the most 15 additional amino acidresidues are replaced; in another embodiment, at the most 10 amino acidresidues are replaced; in another embodiment, at the most 5 amino acidresidues are replaced.

In another embodiment of the invention the Leu residue in position 305or the Phe residue in position 374 of SEQ ID NO 1 and at least oneresidue in position 274 and/or 300-304 and/or position 306-312 have beenreplaced.

In another embodiment, the Leu residue in position 305 or the Pheresidue in position 374 of SEQ ID NO 1 and at least the residue inposition 274 have been replaced.

In another embodiment, the Leu residue in position 305 or the Pheresidue in position 374 of SEQ ID NO 1 and at least one residue inposition 300-304 have been replaced.

In another embodiment, the Leu residue in position 305 or the Pheresidue in position 374 of SEQ ID NO 1 and at least one residue inposition 306-312 have been replaced.

In another embodiment, the Ala residue in position 274 has been replacedby Met or Leu or Lys or Arg; and/or the Arg residue in position 304 hasbeen replaced by Tyr or Phe or Leu or Met; and/or the Met residue inposition 306 has been replaced by Asp or Asn; and/or the Asp residue inposition 309 has been replaced by Ser or Thr.

In another embodiment, the Leu residue in position 305 or the Pheresidue in position 374 is the only amino acid residue that has beenreplaced.

In one embodiment, the Leu residue in position 305 has been replaced. Inanother embodiment, the Phe residue in position 374 has been replaced.

In one embodiment, the Phe residue in position 374 is the only aminoacid residue that has been replaced.

In another embodiment, the Leu residue in position 305 is the only aminoacid residue that has been replaced.

In a specific embodiment, the Leu residue in position 305 has beenreplaced by Val.

In another embodiment, the Leu residue in position 305 has been replacedby an amino acid residue selected from the group consisting of Val, Tyrand Ile, or the Phe residue in position 374 has been replaced by Pro.

In one embodiment of the invention the residues 300-322, 305-322,300-312, or 305-312 of SEQ ID NO 1 are replaced by the correspondingsequences from trypsin (SEQ ID NOS 3, 7, 11, 15, respectively), thrombin(SEQ ID NOS 4, 8, 12, 16, respectively), Factor Xa (SEQ ID NO 5, 9, 13,17, respectively) or another constitutively active serine protease. Inyet another embodiment, one or more of residues 313-322 of SEQ ID NO 1is/are deleted.

In one aspect, the amino acid residue at position 305 has been replacedby an amino acid residue selected from a list of Ala, Val, Ile, Met,Phe, Trp, Pro, Gly, Ser, Thr, Cys, Tyr, Asn, Glu, Lys, Arg, His, Asp andGln, or the amino acid residue at position 374 has been replaced by anamino acid residue selected from a list of Ala, Val, Leu, Ile, Met, Trp,Pro, Gly, Ser, Thr, Cys, Tyr, Asn, Glu, Lys, Arg, His, Asp or Gln, withthe proviso that the variant is not FVII(Ala305).

In another aspect, the amino acid residue at position 305 has beenreplaced by an amino acid residue selected from a list of Ala, Val, Ile,Met, Phe, Trp, Pro, Gly, Ser, Thr, Cys, Tyr, Asn, Glu, Lys, Arg, His,Asp and Gln, or the amino acid residue at position 374 has been replacedby an amino acid residue selected from a list of Ala, Val, Leu, Ile,Met, Trp, Pro, Gly, Ser, Thr, Cys, Tyr, Asn, Glu, Lys, Arg, His, Asp orGln.

In another aspect, the amino acid residue at position 305 has beenreplaced by an amino acid residue that can be encoded by nucleic acids,such as Ala, Val, Ile, Met, Phe, Trp, Pro, Gly, Ser, Thr, Cys, Tyr, Asn,Glu, Lys, Arg, His, Asp and Gln, and the amino acid residue at position374 has been replaced by an amino acid residue that can be encoded bynucleic acid, such as Ala, Val, Leu, Ile, Met, Trp, Pro, Gly, Ser, Thr,Cys, Tyr, Asn, Glu, Lys, Arg, His, Asp or Gln.

In one embodiment, the amino acid residue at position 305 has beenreplaced by an amino acid residue selected from a list of Ala, Val, Ile,Met, Phe, Trp, Pro, Gly, Ser, Thr, Cys, Tyr, Asn, Glu, Lys, Arg, His,Asp and Gln, and the amino acid residue at position 374 has beenreplaced by an amino acid residue selected from a list of Ala, Val, Leu,Ile, Met, Trp, Pro, Gly, Ser, Thr, Cys, Tyr, Asn, Glu, Lys, Arg, His,Asp or Gln.

The present invention also provides a human coagulation Factor VIIvariant, wherein the ratio between the activity of the variant and theactivity of the native Factor VII polypeptide shown in SEQ ID NO 1 is atleast about 1.25 when tested in the “In Vitro Hydrolysis Assay” definedherein. In one embodiment, the ratio is at least about 2.0; in yetanother embodiment, at least about 4.0.

In another aspect, the invention provides human coagulation Factor VIIavariants that have increased tissue factor-independent activity comparedto native human coagulation Factor VIIa. In another aspect, theincreased activity is not accompanied by changes in the substratespecificity. In another aspect of the invention, the binding of thevariants to tissue factor should not be impaired and the variants shouldhave at least the activity of wild-type Factor VIIa when bound to tissuefactor.

Another aspect of the present invention relates to a nucleic acidconstruct, preferably a DNA construct, comprising a nucleotide sequenceencoding a Factor VII variant according to the invention.

In another aspect, the invention provides a recombinant vectorcomprising the nucleic acid construct.

Another aspect of the present invention relates to a recombinant hostcell, preferably of mammalian origin, comprising the nucleic acidconstruct or the recombinant vector.

In one embodiment, the recombinant host cells are CHO or BHK cells.

Another aspect of the present invention relates to a transgenic animalor a transgenic plant containing and expressing the nucleic acidconstruct.

Other aspects of the present invention relate to a pharmaceuticalcomposition comprising a human coagulation Factor VII variant whereinthe Leu residue in position 305 or the Phe residue in position 374 ofSEQ ID NO 1 has been replaced by another amino acid residue which can beencoded by nucleic acid constructs and, optionally, wherein at least oneother amino acid residue in the remaining positions in the proteasedomain has been replaced by another amino acid residue which can beencoded by nucleic acid constructs, optionally in combination with apharmaceutically acceptable carrier; to the human coagulation Factor VIIvariant for use as a medicament; to the use of the human coagulationFactor VII variant for the preparation of a composition for thetreatment or prophylaxis of bleeding episodes or for the enhancement ofthe normal haemostatic system; to a method for the treatment orprophylaxis of bleeding episodes in a subject or for the enhancement ofthe normal haemostatic system; and to methods for producing a Factor VIIvariant according to the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the full amino acid sequence of native human coagulationFactor VII (SEQ ID NO 1).

FIG. 2 shows the region 300-322 of human coagulation Factor VII and thecorresponding region of homologous serine proteases:

Region 300-322 of Factor VII (SEQ ID NO 2)

Corresponding region of trypsin (SEQ ID NO 3)Corresponding region of thrombin (SEQ ID NO 4)Corresponding region of FXa (SEQ ID NO 5)

Region 305-322 of Factor VII (SEQ ID NO 6)

Corresponding region of trypsin (SEQ ID NO 7)Corresponding region of thrombin (SEQ ID NO 8)Corresponding region of FXa (SEQ ID NO 9)

Region 300-312 of Factor VII (SEQ ID NO 10)

Corresponding region of trypsin (SEQ ID NO 11)Corresponding region of thrombin (SEQ ID NO 12)Corresponding region of FXa (SEQ ID NO 13)

Region 305-312 of Factor VII (SEQ ID NO 14)

Corresponding region of trypsin (SEQ ID NO 15)Corresponding region of thrombin (SEQ ID NO 16)Corresponding region of FXa (SEQ ID NO 17)

DETAILED DESCRIPTION OF THE INVENTION

It has now been found that Factor VIIa variants wherein at least one ofthe amino acid residues Leu305 or Phe374 (and optionally one or moreadditional residues) is/are replaced by another amino acid residue havecoagulant activity.

The residues Leu305 and Phe374 are located at each end of an α-helixstarting at residue 307. This α-helix is found in the tissuefactor-complexed form of Factor VIIa. In free Factor VIIa (Factor VIIanot bound to tissue factor) the helix is distorted and thus possiblyunstable. The helix is believed to be important to the activity. Thevariants according to the present invention attain the activeconformation, which normally has to be induced by tissue factor.

The activity may be due to a stabilisation of the α-helix starting atresidue 307, a reorientation of the helix or some other change inconformation. Replacement of one of the residues Leu305 or Phe374, whichare located at each end of the helix, will induce a reorientation and/orstabilisation of the helix.

Due to the higher inherent activity of the described Factor VIIa variantcompared to native Factor VIIa, a lower dose will be adequate to obtaina functionally adequate concentration at the site of action and thus itwill be possible to administer a lower dose to the subject havingbleeding episodes or needing enhancement of the normal haemostaticsystem.

As discussed briefly above, it has been found by the present inventorsthat by replacing either the Leu residue in position 305 or the Pheresidue in position 374 with another amino acid, Factor VIIa willspontaneously attain a more active conformation that normally has to beinduced by tissue factor. Examples of preferred amino acid residues,which may replace Leu in position 305, include Val, Tyr and Ile.

Thus, it is contemplated by the present inventors that such Factor VIIavariants exhibit an inherent activity which may be therapeuticallyuseful in situations where the procoagulant activity is independent oftissue factor (Factor Xa generation on the platelet surface) such aswhen high doses of, for example, NovoSeven® are administered.

As said, replacement of other amino acid residues in the sequence may,in addition to the effect obtained by replacement of the Leu305 or thePhe374 residue, further facilitate formation of the active conformationof the molecule. In principle these remaining positions may be anywhere(except, of course, in position 305 or 374) in the protease domain. Itis believed, however, that the most pronounced effects will be seen whenthe above-mentioned mutations are carried out in the vicinity(sequential or three-dimensional) of residue 305 (or 374).

It is well established that replacement of a few amino acid residues inthe N-terminal Gla domain (residues 1-37) of Factor VII can provide theprotein with a substantially higher affinity for membrane phospholipids,such as membrane phospholipids of tissue factor-bearing cells or ofplatelets, thereby generating Factor VII derivatives which have animproved procoagulant effect.

Thus, the Factor VII variants mentioned above may, in addition to thealready performed amino acid replacement in positions 305 or 374 and theoptional amino acid replacements in positions 274, 300-304 and 306-310or elsewhere in the protease domain, also have some amino acid residuesreplaced in the N-terminal Gla domain, thereby obtaining a proteinhaving an increased activity as well as an increased affinity formembrane phospholipids compared to native Factor VII.

Preferably the amino acid residues in positions 10 and 32 (referring toSEQ ID NO 1) of Factor VII may be replaced with another amino acidresidue that can be encoded by nucleic acid constructs.

Examples of preferred amino acid residues to be incorporated in theabove-mentioned positions are:

The amino acid residue Pro in position 10 is replaced by Gln, Arg, His,Gln, Asn or Lys; and/or the amino acid residue Lys in position 32 isreplaced by Glu, Gln or Asn.

Other residues in the Gla domain, based on the different phospholipidaffinities and sequences of the vitamin K-dependent plasma proteins, mayalso be considered for substitution.

In the present context the three-letter or one-letter indications of theamino acids have been used in their conventional meaning as indicated intable 1. Unless indicated explicitly, the amino acids mentioned hereinare L-amino acids. Further, the left and right ends of an amino acidsequence of a peptide are, respectively, the N- and C-termini unlessotherwise specified.

TABLE 1 Abbreviations for amino acids: Amino acid Tree-letter codeOne-letter code Glycine Gly G Proline Pro P Alanine Ala A Valine Val VLeucine Leu L Isoleucine Ile I Methionine Met M Cysteine Cys CPhenylalanine Phe F Tyrosine Tyr Y Tryptophan Trp W Histidine His HLysine Lys K Arginine Arg R Glutamine Gln Q Asparagine Asn N GlutamicAcid Glu E Aspartic Acid Asp D

The term “N-terminal GLA-domain” means the amino acid sequence 1-37 ofFactor VII.

The term “protease domain” means the amino acid sequence 153-406 ofFactor VII (the heavy-chain of Factor VIIa).

The three-letter indication “GLA” means 4-carboxyglutamic acid(γ-carboxyglutamate).

The indication “FVII(Ala305)” means Factor VII as shown in SEQ ID NO 1wherein the Leu residue in position 305 has been replaced by Ala.

The term “Factor VII” or “FVII” as used herein is intended to comprisethe inactive one-chain zymogen Factor VII molecule as well as theactivated two-chain Factor VII molecule, and may, where appropriate, beused interchangeably with the terms “polypeptide”, “protein”, “protease”and “enzyme”.

As used herein the term “nucleic acid construct” is intended to mean anynucleic acid molecule of cDNA, genomic DNA, synthetic DNA or RNA origin.The term “construct” is intended to indicate a nucleic acid segmentwhich may be single- or double-stranded, and which may be based on acomplete or partial naturally occurring nucleotide sequence encoding thepolypeptide of interest. The construct may optionally contain othernucleic acid segments. In a similar way, the term “amino acid residuewhich can be encoded by nucleic acid constructs” covers amino acidresidues which can be encoded by the nucleic acid constructs definedabove, i.e. amino acids such as Ala, Val, Leu, Ile, Met, Phe, Trp, Pro,Gly, Ser, Thr, Cys, Tyr, Asn, Glu, Lys, Arg, His, Asp and Gln.

In the present context, the term “treatment” is meant to include bothprevention of an expected bleeding, such as in surgery, and regulationof an already occurring bleeding, such as in trauma, with the purpose ofinhibiting or minimising the bleeding. Prophylactic administration ofthe Factor VIIa variant according to the invention is thus included inthe term “treatment”.

The term “activity” means the ability to generate thrombin, the term“inherent activity” also includes the ability to generate thrombin onthe surface of activated platelets in the absence of tissue factor.

The term “enhancement of the normal haemostatic system” means anenhancement of the ability to generate thrombin.

As used herein the term “bleeding disorder” reflects any defect,congenital, acquired or induced, of cellular or molecular origin that ismanifested in bleedings. Examples are clotting factor deficiencies (e.g.haemophilia A and B or deficiency of coagulation Factors XI or VII),clotting factor inhibitors, defective platelet function,thrombocytopenia or von Willebrand's disease.

The term “bleeding episodes” is meant to include uncontrolled andexcessive bleeding which is a major problem both in connection withsurgery and other forms of tissue damage. Uncontrolled and excessivebleeding may occur in subjects having a normal coagulation system andsubjects having coagulation or bleeding disorders. Clotting factordeficiencies (haemophilia A and B, deficiency of coagulation factors XIor VII) or clotting factor inhibitors may be the cause of bleedingdisorders. Excessive bleedings also occur in subjects with a normallyfunctioning blood clotting cascade (no clotting factor deficiencies or-inhibitors against any of the coagulation factors) and may be caused bya defective platelet function, thrombocytopenia or von Willebrand'sdisease. In such cases, the bleedings may be likened to those bleedingscaused by haemophilia because the haemostatic system, as in haemophilia,lacks or has abnormal essential clotting “compounds” (such as plateletsor von Willebrand factor protein) that causes major bleedings. Insubjects who experience extensive tissue damage in association withsurgery or vast trauma, the normal haemostatic mechanism may beoverwhelmed by the demand of immediate haemostasis and they may developbleeding in spite of a normal haemostatic mechanism. Achievingsatisfactory haemostasis also is a problem when bleedings occur inorgans such as the brain, inner ear region and eyes with limitedpossibility for surgical haemostasis. The same problem may arise in theprocess of taking biopsies from various organs (liver, lung, tumourtissue, gastrointestinal tract) as well as in laparoscopic surgery.Common for all these situations is the difficulty to provide haemostasisby surgical techniques (sutures, clips, etc.) which also is the casewhen bleeding is diffuse (haemorrhagic gastritis and profuse uterinebleeding). Acute and profuse bleedings may also occur in subjects onanticoagulant therapy in whom a defective haemostasis has been inducedby the therapy given. Such subjects may need surgical interventions incase the anticoagulant effect has to be counteracted rapidly. Radicalretropubic prostatectomy is a commonly performed procedure for subjectswith localized prostate cancer. The operation is frequently complicatedby significant and sometimes massive blood loss. The considerable bloodloss during prostatectomy is mainly related to the complicatedanatomical situation, with various densely vascularized sites that arenot easily accessible for surgical haemostasis, and which may result indiffuse bleeding from a large area. Another situation that may causeproblems in the case of unsatisfactory haemostasis is when subjects witha normal haemostatic mechanism are given anticoagulant therapy toprevent thromboembolic disease. Such therapy may include heparin, otherforms of proteoglycans, warfarin or other forms of vitamin K-antagonistsas well as aspirin and other platelet aggregation inhibitors.

In one embodiment of the invention, the bleeding is associated withhaemophilia. In another embodiment, the bleeding is associated withhaemophilia with acquired inhibitors. In another embodiment, thebleeding is associated with thrombocytopenia. In another embodiment, thebleeding is associated with von Willebrand's disease. In anotherembodiment, the bleeding is associated with severe tissue damage. Inanother embodiment, the bleeding is associated with severe trauma. Inanother embodiment, the bleeding is associated with surgery. In anotherembodiment, the bleeding is associated with laparoscopic surgery. Inanother embodiment, the bleeding is associated with haemorrhagicgastritis. In another embodiment, the bleeding is profuse uterinebleeding. In another embodiment, the bleeding is occurring in organswith a limited possibility for mechanical haemostasis. In anotherembodiment, the bleeding is occurring in the brain, inner ear region oreyes. In another embodiment, the bleeding is associated with the processof taking biopsies. In another embodiment, the bleeding is associatedwith anticoagulant therapy.

The term “subject” as used herein is intended to mean any animal, inparticular mammals, such as humans, and may, where appropriate, be usedinterchangeably with the term “patient”.

As used herein the term “appropriate growth medium” means a mediumcontaining nutrients and other components required for the growth ofcells and the expression of the nucleic acid sequence encoding theFactor VII variant of the invention.

Preparation of Factor VII Variants

The Factor VII variants described herein may be produced by means ofrecombinant nucleic acid techniques. In general, a cloned wild-typeFactor VII nucleic acid sequence is modified to encode the desiredprotein. This modified sequence is then inserted into an expressionvector, which is in turn transformed or transfected into host cells.Higher eukaryotic cells, in particular cultured mammalian cells, arepreferred as host cells. The complete nucleotide and amino acidsequences for human Factor VII are known (see U.S. Pat. No. 4,784,950,where the cloning and expression of recombinant human Factor VII isdescribed). The bovine Factor VII sequence is described in Takeya etal., J. Biol. Chem. 263:14868-14872 (1988)).

The amino acid sequence alterations may be accomplished by a variety oftechniques. Modification of the nucleic acid sequence may be bysite-specific mutagenesis. Techniques for site-specific mutagenesis arewell known in the art and are described in, for example, Zoller andSmith (DNA 3:479-488, 1984) or “Splicing by extension overlap”, Hortonet al., Gene 77, 1989, pp. 61-68. Thus, using the nucleotide and aminoacid sequences of Factor VII, one may introduce the alteration(s) ofchoice. Likewise, procedures for preparing a DNA construct usingpolymerase chain reaction using specific primers are well known topersons skilled in the art (cf. PCR Protocols, 1990, Academic Press, SanDiego, Calif., USA).

The nucleic acid construct encoding the Factor VII variant of theinvention may suitably be of genomic or cDNA origin, for instanceobtained by preparing a genomic or cDNA library and screening for DNAsequences coding for all or part of the polypeptide by hybridizationusing synthetic oligonucleotide probes in accordance with standardtechniques (cf. Sambrook et al., Molecular Cloning: A Laboratory Manual,2nd. Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989).

The nucleic acid construct encoding the Factor VII variant may also beprepared synthetically by established standard methods, e.g. thephosphoramidite method described by Beaucage and Caruthers, TetrahedronLetters 22 (1981), 1859-1869, or the method described by Matthes et al.,EMBO Journal 3 (1984), 801-805. According to the phosphoramidite method,oligonucleotides are synthesised, e.g. in an automatic DNA synthesiser,purified, annealed, ligated and cloned in suitable vectors.

Furthermore, the nucleic acid construct may be of mixed synthetic andgenomic, mixed synthetic and cDNA or mixed genomic and cDNA originprepared by ligating fragments of synthetic, genomic or cDNA origin (asappropriate), the fragments corresponding to various parts of the entirenucleic acid construct, in accordance with standard techniques.

The nucleic acid construct is preferably a DNA construct,

DNA sequences for use in producing Factor VII variants according to thepresent invention will typically encode a pre-pro polypeptide at theamino-terminus of Factor VII to obtain proper posttranslationalprocessing (e.g. gamma-carboxylation of glutamic acid residues) andsecretion from the host cell. The pre-pro polypeptide may be that ofFactor VII or another vitamin K-dependent plasma protein, such as FactorIX, Factor X, prothrombin, protein C or protein S. As will beappreciated by those skilled in the art, additional modifications can bemade in the amino acid sequence of the Factor VII variants where thosemodifications do not significantly impair the ability of the protein toact as a coagulant. For example, the Factor VII variants can also bemodified in the activation cleavage site to inhibit the conversion ofzymogen Factor VII into its activated two-chain form, as generallydescribed in U.S. Pat. No. 5,288,629.

Expression vectors for use in expressing Factor VIIa variants willcomprise a promoter capable of directing the transcription of a clonedgene or cDNA. Preferred promoters for use in cultured mammalian cellsinclude viral promoters and cellular promoters. Viral promoters includethe SV40 promoter (Subramani et al., Mol. Cell. Biol. 1:854-864, 1981)and the CMV promoter (Boshart et al., Cell 41:521-530, 1985). Aparticularly preferred viral promoter is the major late promoter fromadenovirus 2 (Kaufman and Sharp, Mol. Cell. Biol. 2:1304-1319, 1982).Cellular promoters include the mouse kappa gene promoter (Bergman etal., Proc. Natl. Acad. Sci. USA 81:7041-7045, 1983) and the mouse V_(H)promoter (Loh et al., Cell 33:85-93, 1983). A particularly preferredcellular promoter is the mouse metallothionein-I promoter (Palmiter etal., Science 222:809-814, 1983). Expression vectors may also contain aset of RNA splice sites located downstream from the promoter andupstream from the insertion site for the Factor VII sequence itself.Preferred RNA splice sites may be obtained from adenovirus and/orimmunoglobulin genes. Also contained in the expression vectors is apolyadenylation signal located downstream of the insertion site.Particularly preferred polyadenylation signals include the early or latepolyadenylation signal from SV40 (Kaufman and Sharp, ibid.), thepolyadenylation signal from the adenovirus 5 Elb region, the humangrowth hormone gene terminator (DeNoto et al. Nucl. Acids Res.9:3719-3730, 1981) or the polyadenylation signal from the human FactorVII gene or the bovine Factor VII gene. The expression vectors may alsoinclude a noncoding viral leader sequence, such as the adenovirus 2tripartite leader, located between the promoter and the RNA splicesites; and enhancer sequences, such as the SV40 enhancer.

Cloned DNA sequences are introduced into cultured mammalian cells by,for example, calcium phosphate-mediated transfection (Wigler et al.,Cell 14:725-732, 1978; Corsaro and Pearson, Somatic Cell Genetics7:603-616, 1981; Graham and Van der Eb, Virology 52d:456-467, 1973) orelectroporation (Neumann et al., EMBO J. 1:841-845, 1982). To identifyand select cells that express the exogenous DNA, a gene that confers aselectable phenotype (a selectable marker) is generally introduced intocells along with the gene or cDNA of interest. Preferred selectablemarkers include genes that confer resistance to drugs such as neomycin,hygromycin, and methotrexate. The selectable marker may be anamplifiable selectable marker. A preferred amplifiable selectable markeris a dihydrofolate reductase (DHFR) sequence. Selectable markers arereviewed by Thilly (Mammalian Cell Technology Butterworth Publishers,Stoneham, Mass., incorporated herein by reference). The person skilledin the art will easily be able to choose suitable selectable markers.

Selectable markers may be introduced into the cell on a separate plasmidat the same time as the gene of interest, or they may be introduced onthe same plasmid. If, on the same plasmid, the selectable marker and thegene of interest may be under the control of different promoters or thesame promoter, the latter arrangement producing a dicistronic message.Constructs of this type are known in the art (for example, Levinson andSimonsen, U.S. Pat. No. 4,713,339). It may also be advantageous to addadditional DNA, known as “carrier DNA,” to the mixture that isintroduced into the cells.

After the cells have taken up the DNA, they are grown in an appropriategrowth medium, typically for 1-2 days, to begin expressing the gene ofinterest. The medium used to culture the cells may be any conventionalmedium suitable for growing the host cells, such as minimal or complexmedia containing appropriate supplements. Suitable media are availablefrom commercial suppliers or may be prepared according to publishedrecipes (e.g. in catalogues of the American Type Culture Collection).The media are prepared using procedures known in the art (see, e.g.,references for bacteria and yeast; Bennett, J. W. and LaSure, L.,editors, More Gene Manipulations in Fungi, Academic Press, CA, 1991).Growth media generally include a carbon source, a nitrogen source,essential amino acids, essential sugars, vitamins, salts, phospholipids,proteins and growth factors. For production of gamma-carboxylated FactorVII variants, the medium will contain vitamin K, preferably at aconcentration of about 0.1 mg/ml to about 5 mg/ml. Drug selection isthen applied to select for the growth of cells that are expressing theselectable marker in a stable fashion. For cells that have beentransfected with an amplifiable selectable marker the drug concentrationmay be increased to select for an increased copy number of the clonedsequences, thereby increasing expression levels. Clones of stablytransfected cells are then screened for expression of the desired FactorVII variant.

Preferred mammalian cell lines include the CHO (ATCC CCL 61), COS-1(ATCC CRL 1650), baby hamster kidney (BHK) and 293 (ATCC CRL 1573;Graham et al., J. Gen. Virol. 36:59-72, 1977) cell lines. A preferredBHK cell line is the tk⁻ ts13 BHK cell line (Waechter and Baserga, Proc.Natl. Acad. Sci. USA 79:1106-1110, 1982), hereinafter referred to as BHK570 cells. The BHK 570 cell line is available from the American TypeCulture Collection, 12301 Parklawn Dr., Rockville, Md. 20852, under ATCCaccession number CRL 10314. A tk⁻ ts13 BHK cell line is also availablefrom the ATCC under accession number CRL 1632. In addition, a number ofother cell lines may be used, including Rat Hep I (Rat hepatoma; ATCCCRL 1600), Rat Hep II (Rat hepatoma; ATCC CRL 1548), TCMK (ATCC CCL139), Human lung (ATCC HB 8065), NCTC 1469 (ATCC CCL 9.1) and DUKX cells(Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216-4220, 1980).

Transgenic animal technology may be employed to produce the Factor VIIvariants of the invention. It is preferred to produce the proteinswithin the mammary glands of a host female mammal. Expression in themammary gland and subsequent secretion of the protein of interest intothe milk overcomes many difficulties encountered in isolating proteinsfrom other sources. Milk is readily collected, available in largequantities, and biochemically well characterized. Furthermore, the majormilk proteins are present in milk at high concentrations (typically fromabout 1 to 15 g/l).

From a commercial point of view, it is clearly preferable to use as thehost a species that has a large milk yield. While smaller animals suchas mice and rats can be used (and are preferred at the proof ofprinciple stage), it is preferred to use livestock mammals including,but not limited to, pigs, goats, sheep and cattle. Sheep areparticularly preferred due to such factors as the previous history oftransgenesis in this species, milk yield, cost and the readyavailability of equipment for collecting sheep milk (see, for example,WO 88/00239 for a comparison of factors influencing the choice of hostspecies). It is generally desirable to select a breed of host animalthat has been bred for dairy use, such as East Friesland sheep, or tointroduce dairy stock by breeding of the transgenic line at a laterdate. In any event, animals of known, good health status should be used.

To obtain expression in the mammary gland, a transcription promoter froma milk protein gene is used. Milk protein genes include those genesencoding caseins (see U.S. Pat. No. 5,304,489), beta-lactoglobulin,a-lactalbumin, and whey acidic protein. The beta-lactoglobulin (BLG)promoter is preferred. In the case of the ovine beta-lactoglobulin gene,a region of at least the proximal 406 by of 5′ flanking sequence of thegene will generally be used, although larger portions of the 5′ flankingsequence, up to about 5 kbp, are preferred, such as a ˜4.25 kbp DNAsegment encompassing the 5′ flanking promoter and non-coding portion ofthe beta-lactoglobulin gene (see Whitelaw et al., Biochem. J. 286: 31-39(1992)). Similar fragments of promoter DNA from other species are alsosuitable.

Other regions of the beta-lactoglobulin gene may also be incorporated inconstructs, as may genomic regions of the gene to be expressed. It isgenerally accepted in the art that constructs lacking introns, forexample, express poorly in comparison with those that contain such DNAsequences (see Brinster et al., Proc. Natl. Acad. Sci. USA 85: 836-840(1988); Palmiter et al., Proc. Natl. Acad. Sci. USA 88: 478-482 (1991);Whitelaw et al., Transgenic Res. 1: 3-13 (1991); WO 89/01343; and WO91/02318, each of which is incorporated herein by reference). In thisregard, it is generally preferred, where possible, to use genomicsequences containing all or some of the native introns of a geneencoding the protein or polypeptide of interest, thus the furtherinclusion of at least some introns from, e.g, the beta-lactoglobulingene, is preferred. One such region is a DNA segment that provides forintron splicing and RNA polyadenylation from the 3′ non-coding region ofthe ovine beta-lactoglobulin gene. When substituted for the natural 3′non-coding sequences of a gene, this ovine beta-lactoglobulin segmentcan both enhance and stabilize expression levels of the protein orpolypeptide of interest. Within other embodiments, the regionsurrounding the initiation ATG of the variant Factor VII sequence isreplaced with corresponding sequences from a milk specific protein gene.Such replacement provides a putative tissue-specific initiationenvironment to enhance expression. It is convenient to replace theentire variant Factor VII pre-pro and 5′ non-coding sequences with thoseof, for example, the BLG gene, although smaller regions may be replaced.

For expression of Factor VII variants in transgenic animals, a DNAsegment encoding variant Factor VII is operably linked to additional DNAsegments required for its expression to produce expression units. Suchadditional segments include the above-mentioned promoter, as well assequences that provide for termination of transcription andpolyadenylation of mRNA. The expression units will further include a DNAsegment encoding a secretory signal sequence operably linked to thesegment encoding modified Factor VII. The secretory signal sequence maybe a native Factor VII secretory signal sequence or may be that ofanother protein, such as a milk protein (see, for example, von Heijne,Nucl. Acids Res. 14: 4683-4690 (1986); and Meade et al., U.S. Pat. No.4,873,316, which are incorporated herein by reference).

Construction of expression units for use in transgenic animals isconveniently carried out by inserting a variant Factor VII sequence intoa plasmid or phage vector containing the additional DNA segments,although the expression unit may be constructed by essentially anysequence of ligations. It is particularly convenient to provide a vectorcontaining a DNA segment encoding a milk protein and to replace thecoding sequence for the milk protein with that of a variant Factor VIIpolypeptide; thereby creating a gene fusion that includes the expressioncontrol sequences of the milk protein gene. In any event, cloning of theexpression units in plasmids or other vectors facilitates theamplification of the variant Factor VII sequence. Amplification isconveniently carried out in bacterial (e.g. E. coli) host cells, thusthe vectors will typically include an origin of replication and aselectable marker functional in bacterial host cells. The expressionunit is then introduced into fertilized eggs (including early-stageembryos) of the chosen host species. Introduction of heterologous DNAcan be accomplished by one of several routes, including microinjection(e.g. U.S. Pat. No. 4,873,191), retroviral infection (Jaenisch, Science240: 1468-1474 (1988)) or site-directed integration using embryonic stem(ES) cells (reviewed by Bradley et al. Bio/Technology 10: 534-539(1992)). The eggs are then implanted into the oviducts or uteri ofpseudopregnant females and allowed to develop to term. Offspringcarrying the introduced DNA in their germ line can pass the DNA on totheir progeny in the normal, Mendelian fashion, allowing the developmentof transgenic herds. General procedures for producing transgenic animalsare known in the art (see, for example, Hogan et al., Manipulating theMouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory, 1986;Simons et al., Bio/Technology 6: 179-183 (1988); Wall et al., Biol.Reprod. 32: 645-651 (1985); Buhler et al., Bio/Technology 8: 140-143(1990); Ebert et al., Bio/Technology 9: 835-838 (1991); Krimpenfort etal., Bio/Technology 9: 844-847 (1991); Wall et al. J. Cell. Biochem. 49:113-120 (1992); U.S. Pat. No. 4,873,191; U.S. Pat. No. 4,873,316; WO88/00239, WO 90/05188, WO 92/11757; and GB 87/00458). Techniques forintroducing foreign DNA sequences into mammals and their germ cells wereoriginally developed in the mouse (see, e.g., Gordon et al., Proc. Natl.Acad. Sci. USA 77: 7380-7384 (1980); Gordon and Ruddle, Science 214:1244-1246 (1981); Palmiter and Brinster, Cell 41: 343-345 (1985);Brinster et al. Proc. Natl. Acad. Sci. USA 82: 4438-4442 (1985); andHogan et al. (ibid.)). These techniques were subsequently adapted foruse with larger animals, including livestock species (see, e.g., WO88/00239, WO 90/05188, and WO 92/11757; and Simons et al.,Bio/Technology 6: 179-183 (1988)). To summarise, in the most efficientroute used to date in the generation of transgenic mice or livestock,several hundred linear molecules of the DNA of interest are injectedinto one of the pro-nuclei of a fertilized egg according to establishedtechniques. Injection of DNA into the cytoplasm of a zygote can also beemployed.

Production in transgenic plants may also be employed. Expression may begeneralised or directed to a particular organ, such as a tuber (see,Hiatt, Nature 344:469-479 (1990); Edelbaum et al., J. Interferon Res.12:449-453 (1992); Sijmons et al. Bio/Technology 8:217-221 (1990); andEP 0 255 378).

The Factor VII variants of the invention are recovered from cell culturemedium or milk. The Factor VII variants of the present invention may bepurified by a variety of procedures known in the art including, but notlimited to, chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing (IEF), differential solubility (e.g.,ammonium sulfate precipitation), or extraction (see, e.g., ProteinPurification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, NewYork, 1989). Preferably, they may be purified by affinity chromatographyon an anti-Factor VII antibody column. The use of calcium-dependentmonoclonal antibodies, as described by Wakabayashi et al., J. Biol.Chem. 261:11097-11108, (1986) and Thim et al., Biochemistry 27:7785-7793, (1988), is particularly preferred. Additional purificationmay be achieved by conventional chemical purification means, such ashigh performance liquid chromatography. Other methods of purification,including barium citrate precipitation, are known in the art, and may beapplied to the purification of the novel Factor VII variants describedherein (see, for example, Scopes, R., Protein Purification,Springer-Verlag, N.Y., 1982).

For therapeutic purposes it is preferred that the Factor VII variants ofthe invention are substantially pure. Thus, in a preferred embodiment ofthe invention the Factor VII variants of the invention is purified to atleast about 90 to 95% homogeneity, preferably to at least about 98%homogeneity. Purity may be assessed by e.g. gel electrophoresis andamino-terminal amino acid sequencing.

The Factor VII variant is cleaved at its activation site in order toconvert it to its two-chain form. Activation may be carried outaccording to procedures known in the art, such as those disclosed byOsterud, et al., Biochemistry 11:2853-2857 (1972); Thomas, U.S. Pat. No.4,456,591; Hedner and Kisiel, J. Clin. Invest. 71:1836-1841 (1983); orKisiel and Fujikawa, Behring Inst. Mitt. 73:29-42 (1983). Alternatively,as described by Bjoern et al. (Research Disclosure, 269 September 1986,pp. 564-565), Factor VII may be activated by passing it through anion-exchange chromatography column, such as Mono Q® (Pharmacia fineChemicals) or the like. The resulting activated Factor VII variant maythen be formulated and administered as described below.

Assays

The invention also provides suitable assays for selecting preferredFactor VIIa variants according to the invention. These assays can beperformed as a simple preliminary in vitro test.

Thus, Example 6 herein discloses a simple test (entitled “In VitroHydrolysis Assay”) for the activity of Factor VIIa variants of theinvention. Based thereon, Factor VIIa variants which are of particularinterest are such variants where the ratio between the activity of thevariant and the activity of native Factor VII shown in FIG. 1 is above1.0, e.g. at least about 1.25, preferably at least about 2.0, such as atleast about 3.0 or, even more preferred, at least about 4.0 when testedin the “In Vitro Hydrolysis Assay” defined herein.

The activity of the variants can also be measured using a physiologicalsubstrate such as factor X (see Example 7), suitably at a concentrationof 100-1000 nM, where the factor Xa generated is measured after theaddition of a suitable chromogenic substrate (eg. S-2765). In addition,the activity assay may be run at physiological temperature.

The ability of the Factor VIIa variants to generate thrombin can also bemeasured in an assay comprising all relevant coagulation factors andinhibitors at physiological concentrations (minus factor VIII whenmimicking hemophilia A conditions) and activated platelets (as describedon p. 543 in Monroe et al. (1997) Brit. J. Haematol. 99, 542-547 whichis hereby incorporated as reference).

Administration and Pharmaceutical Compositions

The Factor VII variants according to the present invention may be usedto control bleeding disorders which have several causes such as clottingfactor deficiencies (e.g. haemophilia A and B or deficiency ofcoagulation factors XI or VII) or clotting factor inhibitors, or theymay be used to control excessive bleeding occurring in subjects with anormally functioning blood clotting cascade (no clotting factordeficiencies or inhibitors against any of the coagulation factors). Thebleedings may be caused by a defective platelet function,thrombocytopenia or von Willebrand's disease. They may also be seen insubjects in whom an increased fibrinolytic activity has been induced byvarious stimuli.

In subjects who experience extensive tissue damage in association withsurgery or vast trauma, the haemostatic mechanism may be overwhelmed bythe demand of immediate haemostasis and they may develop bleedings inspite of a normal haemostatic mechanism. Achieving satisfactoryhaemostasis is also a problem when bleedings occur in organs such as thebrain, inner ear region and eyes and may also be a problem in cases ofdiffuse bleedings (haemorrhagic gastritis and profuse uterine bleeding)when it is difficult to identify the source. The same problem may arisein the process of taking biopsies from various organs (liver, lung,tumour tissue, gastrointestinal tract) as well as in laparoscopicsurgery. These situations share the difficulty of providing haemostasisby surgical techniques (sutures, clips, etc.). Acute and profusebleedings may also occur in subjects on anticoagulant therapy in whom adefective haemostasis has been induced by the therapy given. Suchsubjects may need surgical interventions in case the anticoagulanteffect has to be counteracted rapidly. Another situation that may causeproblems in the case of unsatisfactory haemostasis is when subjects witha normal haemostatic mechanism are given anticoagulant therapy toprevent thromboembolic disease. Such therapy may include heparin, otherforms of proteoglycans, warfarin or other forms of vitamin K-antagonistsas well as aspirin and other platelet aggregation inhibitors.

A systemic activation of the coagulation cascade may lead todisseminated intravascular coagulation (DIC). However, suchcomplications have not been seen in subjects treated with high doses ofrecombinant Factor VIIa because of a localised haemostatic process ofthe kind induced by the complex formation between Factor VIIa and TFexposed at the site of vessel wall injury. The Factor VII variantsaccording to the invention may thus also be used in their activated formto control such excessive bleedings associated with a normal haemostaticmechanism.

For treatment in connection with deliberate interventions, the FactorVII variants of the invention will typically be administered withinabout 24 hours prior to performing the intervention, and for as much as7 days or more thereafter. Administration as a coagulant can be by avariety of routes as described herein.

The dose of the Factor VII variants ranges from about 0.05 mg to 500mg/day, preferably from about 1 mg to 200 mg/day, and more preferablyfrom about 10 mg to about 175 mg/day for a 70 kg subject as loading andmaintenance doses, depending on the weight of the subject and theseverity of the condition.

The pharmaceutical compositions are primarily intended for parenteraladministration for prophylactic and/or therapeutic treatment.Preferably, the pharmaceutical compositions are administeredparenterally, i.e., intravenously, subcutaneously, or intramuscularly,or it may be administered by continuous or pulsatile infusion. Thecompositions for parenteral administration comprise the Factor VIIvariant of the invention in combination with, preferably dissolved in, apharmaceutically acceptable carrier, preferably an aqueous carrier. Avariety of aqueous carriers may be used, such as water, buffered water,0.4% saline, 0.3% glycine and the like. The Factor VII variants of theinvention can also be formulated into liposome preparations for deliveryor targeting to the sites of injury. Liposome preparations are generallydescribed in, e.g., U.S. Pat. No. 4,837,028, U.S. Pat. No. 4,501,728,and U.S. Pat. No. 4,975,282. The compositions may be sterilised byconventional, well-known sterilisation techniques. The resulting aqueoussolutions may be packaged for use or filtered under aseptic conditionsand lyophilised, the lyophilised preparation being combined with asterile aqueous solution prior to administration. The compositions maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, calciumchloride, etc.

The concentration of Factor VII variant in these formulations can varywidely, i.e., from less than about 0.5% by weight, usually at or atleast about 1% by weight to as much as 15 or 20% by weight and will beselected primarily by fluid volumes, viscosities, etc., in accordancewith the particular mode of administration selected.

Thus, a typical pharmaceutical composition for intravenous infusioncould be made up to contain 250 ml of sterile Ringer's solution and 10mg of the Factor VII variant. Actual methods for preparing parenterallyadministrable compositions will be known or apparent to those skilled inthe art and are described in more detail in, for example Remington'sPharmaceutical Sciences, 18th ed., Mack Publishing Company, Easton, Pa.(1990).

The compositions containing the Factor VII variants of the presentinvention can be administered for prophylactic and/or therapeutictreatments. In therapeutic applications, compositions are administeredto a subject already suffering from a disease, as described above, in anamount sufficient to cure, alleviate or partially arrest the disease andits complications. An amount adequate to accomplish this is defined as“therapeutically effective amount”. As will be understood by the personskilled in the art amounts effective for this purpose will depend on theseverity of the disease or injury as well as the weight and generalstate of the subject. In general, however, the effective amount willrange from about 0.05 mg up to about 500 mg of the Factor VII variantper day for a 70 kg subject, with dosages of from about 1.0 mg to about200 mg of the Factor VII variant per day being more commonly used.

It must be kept in mind that the materials of the present invention maygenerally be employed in serious disease or injury states, that is, lifethreatening or potentially life threatening situations. In such cases,in view of the minimisation of extraneous substances and general lack ofimmunogenicity of human Factor VII variants in humans, it is possibleand may be felt desirable by the treating physician to administer asubstantial excess of these variant Factor VII compositions.

In prophylactic applications, compositions containing the Factor VIIvariant of the invention are administered to a subject susceptible to orotherwise at risk of a disease state or injury to enhance the subject'sown coagulative capability. Such an amount is defined to be a“prophylactically effective dose.” In prophylactic applications, theprecise amounts once again depend on the subject's state of health andweight, but the dose generally ranges from about 0.05 mg to about 500 mgper day for a 70-kilogram subject, more commonly from about 1.0 mg toabout 200 mg per day for a 70-kilogram subject.

Single or multiple administrations of the compositions can be carriedout with dose levels and patterns being selected by the treatingphysician. For ambulatory subjects requiring daily maintenance levels,the Factor VII variants may be administered by continuous infusion usinge.g. a portable pump system.

Local delivery of the Factor VII variant of the present invention, suchas, for example, topical application may be carried out, for example, bymeans of a spray, perfusion, double balloon catheters, stent,incorporated into vascular grafts or stents, hydrogels used to coatballoon catheters, or other well established methods. In any event, thepharmaceutical compositions should provide a quantity of Factor VIIvariant sufficient to effectively treat the subject.

The present invention is further illustrated by the following exampleswhich, however, are not to be construed as limiting the scope ofprotection. The features disclosed in the foregoing description and inthe following examples may, both separately and in any combinationthereof, be material for realising the invention in diverse formsthereof.

EXAMPLES

The terminology for amino acid substitutions used in the followingexamples are as follows. The first letter represent the amino acidnaturally present at a position of SEQ ID NO 1. The following numberrepresent the position in SEQ ID NO 1. The second letter represent thedifferent amino acid substituting for the natural amino acid. An exampleis [L305V]-FVII, where the leucine at position 305 of SEQ ID NO 1 isreplaced by a valine. In another example, [L305V/M306D/D309S]-FVII, theleucine at position 305 of SEQ ID NO 1 is replaced by a valine and themethionine at position 306 of SEQ ID NO 1 is replaced by an asparticacid and the aspartic acid at position 309 of SEQ ID NO 1 is replaced bya serine, all mutations in the same Factor VII polypeptide.

Example 1 DNA encoding [L305V/M306D/D309S]-FVII, [L305V]-FVII,[L305I]-FVII, [L305I]-FVII and [F374P]-FVII

DNA constructs encoding [L305V/M306D/D309S]-FVII, [L305V]-FVII,[L305I]-FVII, [L305T]-FVII and [F374P]-FVII were prepared bysite-directed mutagenesis using a supercoiled, double stranded DNAvector with an insert of interest and two synthetic primers containingthe desired mutation. The following primers were used:

For [L305V]-FVII: (SEQ ID NO 18) 5′-CGT GCC CCG GGT GAT GAC CCA GGA C-3′(SEQ ID NO 19) 5′-GTC CTG GGT CAT CAC CCG GGG CAC G-3′ For[M306D/D309S]-FVII: (SEQ ID NO 20) 5′-TCT AGA TAC CCA GTC TTG CCT GCAGCA GTC ACG GAA-3′ (SEQ ID NO 21) 5′-TTC CGT GAC TGC TGC AGG CAA GAC TGGGTA TCT AGA-3′ For [F374P]-FVII: (SEQ ID NO 22) 5′-CCG TGG GCC ACC CTGGGG TGT ACA CC-3′ (SEQ ID NO 23) 5′-GGT GTA CAC CCC AGG GTG GCC CACGG-3′ For [L305I]-FVII: (SEQ ID NO 24) 5′-CCT CAA CGT GCC CCG GAT CATGAC CCA GGA C-3′ (SEQ ID NO 25) 5′-GTC CTG GGT CAT GAT CCG GGG CAC GTTGAG G-3′ For [L305T]-FVII: (SEQ ID NO 26) 5′-CCT CAA CGT GCC CCG GAC GATGAC CCA GGA C-3′ (SEQ ID NO 27) 5′-GTC CTG GGT CAT CGT CCG GGG CAC GTTGAG G-3′The oligonucleotide primers, each complementary to opposite strands ofthe vector, were extended during temperature cycling by means of Pfu DNApolymerase. On incorporation of the primers, a mutated plasmidcontaining staggered nicks was generated. Following temperature cycling,the product was treated with DpnI which is specific for methylated andhemimethylated DNA to digest the parental DNA template and to select formutation-containing synthesized DNA.

Procedures for preparing a DNA construct using polymerase chain reactionusing specific primers are well known to persons skilled in the art (cf.PCR Protocols, 1990, Academic Press, San Diego, Calif., USA).

Example 2 Preparation of [L305V/M306D/D309S]-FVII

BHK cells were transfected essentially as previously described (Thim etal. (1988) Biochemistry 27, 7785-7793; Persson and Nielsen (1996) FEBSLett. 385, 241-243) to obtain expression of the variant[L305V/M306D/D309S]-FVII. The Factor VII variant was purified asfollows:

Conditioned medium was loaded onto a 25-ml column of Q Sepharose FastFlow (Pharmacia Biotech) after addition of 5 mM EDTA, 0.1% Triton X-100and 10 mM Tris, adjustment of pH to 8.0 and adjustment of theconductivity to 10-11 mS/cm by adding water. Elution of the protein wasaccomplished by a gradient from 10 mM Tris, 50 mM NaCl, 0.1% TritonX-100, pH 8.0 to 10 mM Tris, 1 M NaCl, 5 mM CaCl₂, 0.1% Triton X-100, pH7.5. The fractions containing [L305V/M306D/D309S]-FVII were pooled, 10mM CaCl₂ was added, and applied to a 25-ml column containing monoclonalantibody F1A2 (Novo Nordisk, Bagsværd, Denmark) coupled toCNBr-activated Sepharose 4B (Pharmacia Biotech). The column wasequilibrated with 50 mM Hepes, pH 7.5, containing 10 mM CaCl₂, 100 mMNaCl and 0.02% Triton X-100. After washing with equilibration buffer andequilibration buffer containing 2 M NaCl, bound material was eluted withequilibration buffer containing 10 mM EDTA instead of CaCl₂. Before useor storage, excess CaCl₂ over EDTA was added or [L305V/M306D/D309S]-FVIIwas transferred to a Ca²⁺-containing buffer. The yield of each step wasfollowed by factor VII ELISA measurements and the purified protein wasanalysed by SDS-PAGE.

Example 3 Preparation of [L305V]-FVII

BHK cells were transfected essentially as previously described (Thim etal. (1988) Biochemistry 27, 7785-7793; Persson and Nielsen (1996) FEBSLett. 385, 241-243) to obtain expression of the variant [L305V]-FVII.The Factor VII variant was purified as follows:

Conditioned medium was loaded onto a 25-ml column of Q Sepharose FastFlow (Pharmacia Biotech) after addition of 5 mM EDTA, 0.1% Triton X-100and 10 mM Tris, adjustment of pH to 8.0 and adjustment of theconductivity to 10-11 mS/cm by adding water. Elution of the protein wasaccomplished by a gradient from 10 mM Tris, 50 mM NaCl, 0.1% TritonX-100, pH 8.0 to 10 mM Tris, 1 M NaCl, 5 mM CaCl₂, 0.1% Triton X-100, pH7.5. The fractions containing [L305V]-FVII were pooled, 10 mM CaCl₂ wasadded, and applied to a 25-ml column containing monoclonal antibody F1A2(Novo Nordisk, Bagsværd, Denmark) coupled to CNBr-activated Sepharose 4B(Pharmacia Biotech). The column was equilibrated with 50 mM Hepes, pH7.5, containing 10 mM CaCl₂, 100 mM NaCl and 0.02% Triton X-100. Afterwashing with equilibration buffer and equilibration buffer containing 2M NaCl, bound material was eluted with equilibration buffer containing10 mM EDTA instead of CaCl₂. Before use or storage, excess CaCl₂ overEDTA was added or [L305V]-FVII was transferred to a Ca²⁺-containingbuffer. The yield of each step was followed by factor VII ELISAmeasurements and the purified protein was analysed by SDS-PAGE.

Example 4 Preparation of [F374P]-FVII

BHK cells were transfected essentially as previously described (Thim etal. (1988) Biochemistry 27, 7785-7793; Persson and Nielsen (1996) FEBSLett. 385, 241-243) to obtain expression of the variant [F374P]-FVII.The Factor VII variant was purified as follows:

Conditioned medium was loaded onto a 25-ml column of Q Sepharose FastFlow (Pharmacia Biotech) after addition of 5 mM EDTA, 0.1% Triton X-100and 10 mM Tris, adjustment of pH to 8.0 and adjustment of theconductivity to 10-11 mS/cm by adding water. Elution of the protein wasaccomplished by a gradient from 10 mM Tris, 50 mM NaCl, 0.1% TritonX-100, pH 8.0 to 10 mM Tris, 1 M NaCl, 5 mM CaCl₂, 0.1% Triton X-100, pH7.5. The fractions containing [F374P]-FVII were pooled, 10 mM CaCl₂ wasadded, and applied to a 25-ml column containing monoclonal antibody F1A2(Novo Nordisk, Bagsværd, Denmark) coupled to CNBr-activated Sepharose 4B(Pharmacia Biotech). The column was equilibrated with 50 mM Hepes, pH7.5, containing 10 mM CaCl₂, 100 mM NaCl and 0.02% Triton X-100. Afterwashing with equilibration buffer and equilibration buffer containing 2M NaCl, bound material was eluted with equilibration buffer containing10 mM EDTA instead of CaCl₂. Before use or storage, excess CaCl₂ overEDTA was added or [F374P]-FVII was transferred to a Ca²⁺-containingbuffer. The yield of each step was followed by factor VII ELISAmeasurements and the purified protein was analysed by SDS-PAGE.

Example 5 Preparation of [L305I]-FVII and [L305T]-FVII

BHK cells are transfected essentially as previously described (Thim etal. (1988) Biochemistry 27, 7785-7793; Persson and Nielsen (1996) FEBSLett. 385, 241-243) to obtain expression of the variant [L305I]-FVII or[L305T]-FVII. The Factor VII variant is purified as follows:

Conditioned medium is loaded onto a 25-ml column of Q Sepharose FastFlow (Pharmacia Biotech) after addition of 5 mM EDTA, 0.1% Triton X-100and 10 mM Tris, adjustment of pH to 8.0 and adjustment of theconductivity to 10-11 mS/cm by adding water. Elution of the protein isaccomplished by a gradient from 10 mM Tris, 50 mM NaCl, 0.1% TritonX-100, pH 8.0 to 10 mM Tris, 1 M NaCl, 5 mM CaCl₂, 0.1% Triton X-100, pH7.5. The fractions containing [L305I]-FVII or [L305T]-FVII are pooled,10 mM CaCl₂ is added, and applied to a 25-ml column containingmonoclonal antibody F1A2 (Novo Nordisk, Bagsværd, Denmark) coupled toCNBr-activated Sepharose 4B (Pharmacia Biotech). The column isequilibrated with 50 mM Hepes, pH 7.5, containing 10 mM CaCl₂, 100 mMNaCl and 0.02% Triton X-100. After washing with equilibration buffer andequilibration buffer containing 2 M NaCl, bound material is eluted withequilibration buffer containing 10 mM EDTA instead of CaCl₂. Before useor storage, excess CaCl₂ over EDTA is added or [L305I]-FVII or[L305T]-FVII are transferred to a Ca²⁺-containing buffer. The yield ofeach step is followed by factor VII ELISA measurements and the purifiedprotein is analysed by SDS-PAGE.

Example 6 In Vitro Hydrolysis Assay

Native (wild-type) Factor VIIa and Factor VIIa variant (both hereafterreferred to as “Factor VIIa”) are assayed in parallel to directlycompare their specific activities. The assay is carried out in amicrotiter plate (MaxiSorp, Nunc, Denmark). The chromogenic substrateD-Ile-Pro-Arg-p-nitroanilide (S-2288, Chromogenix, Sweden), finalconcentration 1 mM, is added to Factor VIIa (final concentration 100 nM)in 50 mM Hepes, pH 7.4, containing 0.1 M NaCl, 5 mM CaCl₂ and 1 mg/mlbovine serum albumin. The absorbance at 405 nm is measured continuouslyin a SpectraMax™ 340 plate reader (Molecular Devices, USA). Theabsorbance developed during a 20-minute incubation, after subtraction ofthe absorbance in a blank well containing no enzyme, is used tocalculate the ratio between the activities of variant and wild-typeFactor VIIa:

Ratio=(A _(405 nm) Factor VIIa variant)/(A _(405 nm) Factor VIIawild-type).

Example 7 In Vitro Proteolysis Assay

Native (wild-type) Factor VIIa and Factor VIIa variant (both hereafterreferred to as “Factor VIIa”) are assayed in parallel to directlycompare their specific activities. The assay is carried out in amicrotiter plate (MaxiSorp, Nunc, Denmark). Factor VIIa (10 nM) andFactor X (0.8 microM) in 100 microL 50 mM Hepes, pH 7.4, containing 0.1M NaCl, 5 mM CaCl₂ and 1 mg/ml bovine serum albumin, are incubated for15 min. Factor X cleavage is then stopped by the addition of 50 microL50 mM Hepes, pH 7.4, containing 0.1 M NaCl, 20 mM EDTA and 1 mg/mlbovine serum albumin. The amount of Factor Xa generated is measured byaddition of the chromogenic substrate Z-D-Arg-Gly-Arg-p-nitroanilide(S-2765, Chromogenix, Sweden), final concentration 0.5 mM. Theabsorbance at 405 nm is measured continuously in a SpectraMax™ 340 platereader (Molecular Devices, USA). The absorbance developed during 10minutes, after subtraction of the absorbance in a blank well containingno FVIIa, is used to calculate the ratio between the proteolyticactivities of variant and wild-type Factor VIIa:

Ratio=(A _(405 nm) Factor VIIa variant)/(A _(405 nm) Factor VIIawild-type).

Example 8 Relative Activities of FVIIa Variants Measured in the AssaysDescribed in Examples 6 and 7

Variant Ratio in example 6 Ratio in example 7 L305V/M306D/D309S-FVIIa3.0 ± 0.1 6.3 ± 0.9 L305V-FVIIa 3.2 ± 0.2 3.3 ± 0.2 F374P-FVIIa 1.4 <1wt-FVIIa 1.0 1.0

1. An isolated human coagulation Factor VII variant comprising asubstitution of Phe in position 374 of SEQ ID NO 1 with an amino acidresidue selected from the group consisting of Val, Ile, Met, Phe, Trp,Pro, Gly, Ser, Thr, Cys, Tyr, Asn, Glu, Lys, Arg, His, Asp and Gln.
 2. AFactor VII variant as defined in claim 1, wherein the substituted aminoacid is Pro.
 3. A Factor VII variant as defined in claim 1, furthercomprising a second substitution selected from the group consisting of(i) position 274; (ii) any of positions 300-304; (iii) any of positions306-312; and (iv) combinations of any of the foregoing.
 4. A Factor VIIvariant as defined in claim 3, wherein the second substitution is atposition
 274. 5. A Factor VII variant as defined in claim 3, wherein thesecond substitution is at any of positions 300-304.
 6. A Factor VIIvariant as defined in claim 3, wherein the second substitution is at anyof positions 306-312.
 7. A Factor VII variant as described in claim 1,wherein the Phe residue in position 374 is the only amino acid residuethat has been replaced relative to the sequence of SEQ ID NO:1.
 8. AFactor VII variant as defined in claim 1, wherein the ratio between theactivity of the variant and the activity of native Factor VIIpolypeptide having a sequence shown in SEQ ID NO 1 is at least about1.25 when tested in an in vitro hydrolysis assay.
 9. A Factor VIIvariant as defined in claim 8, wherein the ratio is at least about 2.0,10. A Factor VII variant as defined in claim 9, wherein the ratio is atleast about 4.0.
 11. An isolated nucleic acid construct comprising anucleotide sequence encoding a Factor VII variant as defined in claim 1.12. A recombinant vector comprising a nucleic acid construct as definedin claim
 11. 13. A recombinant host cell comprising a nucleic acidconstruct as defined in claim
 11. 14. A recombinant host cell as definedin claim 13, wherein the cell is of mammalian origin.
 15. A recombinanthost cell as defined in claim 14, wherein the cell is selected from thegroup consisting of CHO cells and BHK cells.
 16. A method for producinga human coagulation Factor VII variant, which comprises (i) cultivatinga cell as defined in claim 13 in an appropriate growth medium underconditions allowing expression of the nucleic acid construct and (ii)recovering the resulting polypeptide from the culture medium.
 17. Apharmaceutical composition comprising (i) a human coagulation Factor VIIvariant as defined in claim 1 and (ii) a pharmaceutically acceptablecarrier or excipient.
 18. A method for the treatment of bleedingepisodes in a subject or for the enhancement of the normal haemostaticsystem, the method comprising administering to a subject in need of suchtreatment a therapeutically or prophylactically effective amount of ahuman coagulation Factor VII variant as defined in claim 1.